PROJECT SUMMARY Inositol phosphates (IPs) are diffusible intracellular messengers (termed the ?IP code?) that perform key functions in the eukaryotic cell, yet the contributions of these molecules to immunity and host defense remains underexplored. Through unbiased approaches, our laboratory has recently uncovered striking new roles for IP molecules at the host-virus interface: (1) during necroptosis, a pro-inflammatory cell death mechanism critical for antiviral immunity and (2) during infection with human rhinoviruses (RV), among the most common infectious agents in human beings. We have found that IP kinase activity that produces the cellular IP signature (i.e., IP3, IP4, IP5, IP6) is critical for both necroptosis and RV infection via apparently distinct mechanisms. We showed that IP molecules can directly control the necroptotic executioner, mixed-lineage kinase like (MLKL), to unleash necrotic cell death. In contrast, we found that RV infection depends on IPs at a stage preceding cell death and independent of MLKL, indicating as yet unidentified host or viral targets for IPs. However, it is currently unknown how the different IPs act to control these processes on a molecular level, how IP kinases precisely collaborate to determine the cellular IP code, and how this code is regulated during infection or cytokine stimulation to mediate immunity. Our central hypothesis is that inositol phosphates and their kinases play important regulatory roles in innate immune responses and during viral infection through interactions with host or viral targets. Here, we will determine the precise mechanisms for engagement of the inositol phosphate code by these fundamental immune processes by employing a robust genetic and biochemical toolkit and combining our expertise in molecular genetics and virology with a collaborative team of leading experts in inositol phosphate biology and biochemistry. Specifically, we will (1) map the genetic and physical interactions of IP species with the necroptotic executioner MLKL in cellular and biochemical assays, and (2) define the molecular mechanism(s) governing regulation of RV infection by the IP code. Together, these studies will provide fundamental insight into regulation of immune defenses and viral pathogenesis. We expect this work to form a foundation for understanding the roles of the IP code in immunity and infection biology and to provide a basis for development of novel methods to enhance anti-microbial and anti- inflammatory therapeutics.